Project Summary PIP2 is a minor phospholipid (PL) and plays a critical role in a variety of cellular functions but the role of PIP2 in atherosclerosis and Nlrp3/ IL-1? inflammasome pathway is not well characterized. I have previously shown that ABCA1 functions as a phosphatidylinositol 4, 5- bisphosphate (PIP2) floppase, transporting PIP2 from the inner to the outer leaflet of the plasma membrane. ABCA1, a cellular cholesterol efflux transporter, plays a major role in preventing atherosclerosis and inflammation by effluxing excess lipids/cholesterol from cells and by blocking pro-inflammatory pathways. Human patients with mutations in Abca1 suffer from premature atherosclerosis and macrophage-specific knockout of ABCA1/G1 in Ldlr KO mice promotes atherosclerosis and plaque inflammation. The role of pro-inflammatory Nlrp3/IL- 1??pathway in atherosclerosis was highlighted by CANTOS trial showing that anti-IL-1? therapy met the primary trial endpoint, a reduction in a composite of heart attack, stroke and cardiovascular death. Recent studies have shown that Gasdermin D (GsdmD), a newly discovered substrate of the inflammasome, binds to PIP2 on the plasma membrane and oligomerize, generating pores for releasing mature IL-1?. The proposed studies will unravel the novel roles of PIP2 in these pathways and may open new windows for therapeutic intervention to prevent cardiovascular disease (CVD). This proposal will further establish PIP2 as a major regulator of cellular cholesterol efflux and identify the PIP2 flippases (P4-type ATPases that transport PIP2 from the outer to the inner leaflet of the plasma membrane) that in turn regulate cholesterol efflux and inflammation. The proposal will identify the role of GsdmD in atherosclerosis, reverse cholesterol transport (RCT), and in reversing the negative effects of inflammation on RCT. The three main goals of this proposal are; 1) to establish PIP2 as a major cellular cholesterol efflux regulator, 2) to identify and characterize the PIP2 flippase and determine the role of P4-type ATPases in cholesterol efflux and inflammation, and 3) to determine the role of Gasdermin D in atherosclerosis and RCT.